ABSTRACT Staphylococcus aureus is one of the most important pathogens responsible for a wide range of illnesses including life-threatening infections. An important number of these infections are caused by methicillin-susceptible S. aureus (MSSA) and in many parts of the world (including the US), MSSA infections are more common than those caused by methicillin-resistant S. aureus (MRSA). Indeed, MSSA represent a major burden of S. aureus infections and are important contributors to mortality. The treatment of MSSA infections relies on the use of ?-lactams. Indeed, the first line of therapy for severe MSSA infections are the isoxazolyl penicillins (e.g., nafcillin, oxacillin). However, recent data suggest that clinical outcomes in MSSA bacteremia are similar in patients treated with nafcillin/oxacillin vs cefazolin, a first generation cephalosporin with activity against MSSA that appears to be less toxic. Moreover, treatment with nafcillin seem to be associated with increased costs, more drug reactions (including hepatotoxicity, interstitial nephritis and neutropenia) and, possibly, higher mortality. Due to these concerns, an important shift in the treatment of MSSA is occurring whereby clinicians are now using cefazolin as first line of therapy for severe MSSA infections. An important concern of using cefazolin and other cephalosporins as primary therapy for these serious infections is the occurrence of the cefazolin inoculum effect (CzIE), defined as a cefazolin minimal inhibitory concentration of > 16 g/ml when a high inoculum (107 CFU/ml) is used. The CzIE has been associated with failures in the treatment of deep-seated MSSA infections and with the production of certain isotypes of the staphylococcal ?-lactamase. However, the molecular basis and the actual clinical impact of this phenomenon are unknown. Our published and preliminary clinical data indicate that the CzIE is an important contributor to clinical outcomes of severe MSSA infections. Our Prelim. Data also provide evidence that the CzIE is mediated, at least in part, by changes in the processing and extracellular release of the ?- lactamase (BlaZ) enzyme and it is influenced by the catalytic activity of the ?-lactamase isotype. Our hypotheses are that, i) the CzIE is primarily mediated by the activity of extracellular ?-lactamase released into the extracellular milieu, ii) the ability to hydrolyze cefazolin effectively depends on the catalytic activity of the enzyme isotype, and iii) a rapid phenotypic test can be developed to identify isolates with the CzIE and help clinicians in the treatment of MSSA infections. In two specific aims, we will define the molecular and biochemical bases of the CzIE exploring the genetic factors influencing this phenomenon and characterizing novel structural and biochemical isoforms of BlaZ identified via our genomic analyses. We will also aim to develop a rapid assay to identify MSSA strains with the CzIE. Our findings are likely to influence the manner by which MSSA infections are treated and will provide the basis to develop novel diagnostic tools to advance the treatment of these life-threatening infections.